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Dual phase - PNA conjugates for the delivery of PNA through the blood brain barrier

a technology of pna and conjugates, which is applied in the direction of animal/human proteins, sugar derivatives, non-active ingredients of pharmaceuticals, etc., can solve the problems of limited therapeutic value, the protection function of the blood-brain barrier becomes, and the nutrient transporters in the bbb can only deliver low molecular weight substrates

Inactive Publication Date: 2005-10-06
LAMENSDORF ITSCHAK +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0014] In one embodiment, the invention further provides...

Problems solved by technology

The protective function of the blood-brain barrier becomes an important problem in the treatment of neurological diseases.
However, nutrient transporters in the BBB can deliver only low molecular weight substrates.
Unfortunately, these strategies can be used only for the delivery of relatively small molecule.
However, due to their low biomembrane permeability and their relatively rapid degradation oligonucleotides are generally considered to be of limited therapeutic value.
PNAs are apolar molecule with poor bioavaliability properties.
Therefore, unmodified / naked PNA molecules pass poorly through the cell membrane and do not have useful therapeutic applications.
Both ODNs and PNAs cannot cross the endothelial cellular membrane of the BBB effectively, thus, preventing the possible use of these technologies in developing drugs for CNS disorders.

Method used

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  • Dual phase - PNA conjugates for the delivery of PNA through the blood brain barrier
  • Dual phase - PNA conjugates for the delivery of PNA through the blood brain barrier
  • Dual phase - PNA conjugates for the delivery of PNA through the blood brain barrier

Examples

Experimental program
Comparison scheme
Effect test

example 1

PC12 Cells

Uptake of Conjugated PNAs into the Neuronal Cell Line PC12:

[0077] Cells were seeded on 96 well dishes coated with poly-1-lysine. Day after seeding medium was replaced with fresh medium without serum containing different concentrations (0.1-1 μM) of PNAs. Following 3 hours incubation medium was removed and cells were washed three times with acid wash solution and fluorescent determined (FIG. 1).

Control =TTT GCT CTT ACT CAT(SEQ ID No. 39)KBP10 =CHK6HC(SEQ ID No. 40)- TTT GCT CTT ACT CAT(SEQ ID No. 39)- THRPPMWSPVWP(SEQ ID No. 41)KBP11 =CHK6HC(SEQ ID No. 40)- TTT GCT CTT ACT CAT -(SEQ ID No. 39)HAIYPRH(SEQ ID No. 41)

As can be seen from FIG. 1, the uptake of either KBP10 and KBP11 into PC12 cells was much higher than the uptake of PNA alone.

Measurement of Cellular Toxicity:

[0078] PC12 cells were incubated with PNA or peptide-PNA conjugates for 48 hours. At the end of the incubation, cell morphology was examined by light microscopy. Medium was replaced with medium cont...

example 2

bEND3 Cell Line BBB Cellular Model

[0079] Uptake of fluorescence labeled PNA (TTT GCT CTT ACT CAT ) (SEQ ID. No. 39) or peptide-PNA to bEND3 (CHK6HC (SEQ ID. No. 40)-TTT GCT CTT ACT CAT-(SEQ ID. No. 39) HAIYPRH (SEQ ID. No. 41).

[0080] bEND3 cells were seeded on a poly-L-ornithine coated 35 mm dish. 24 hours following seeding the cell culture medium was replaced with DMEM containing 10 μM PNA or peptide-PNA. Cells were incubated for 4 hours. Following incubation cells were washed 3 times with PBS and medium was replaced with fresh DMEM cells as observed by confocal microscopy (FIG. 3). As can be clearly seen, the uptake of peptide-PNA is clearly observed whereas uptake of PNA alone is invisible.

example 3

NMB Cell Line

[0081] Uptake of PNA (GCAT) or conjugated peptide PNA (GCAT-THRPPMWSPVWP) (SEQ ID. No. 42) into the human neuronal cell line NMB. Cells were seeded on 96 well dishes coated with poly-1-ornithine. One day after seeding the medium was replaced with a fresh medium without serum, containing 1 micromolar PNAs. Following 15 or 60 min. incubation medium was removed and cells were washed three times with acid wash solution. Fluorescent was determined (FIG. 4).

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Abstract

The invention provides molecule comprising a nucleic acid, a peptide ligand, which binds to a specific receptor and a positively charge peptide moiety with lysosomatic properties, useful in the delivery of a nucleic acid across a cellular membrane. The invention further relates to the use of these compounds for the delivery of a nucleic acid to the brain across the blood brain barrier for diagnostic and therapeutic applications

Description

CROSS REFERENCE DATA [0001] This Application claims the priority of U.S. Provisional Application No. 60 / 510,137 filed Oct. 14, 2003.FIELD OF THE INVENTION [0002] The invention provides compounds comprising a nucleic acid, a peptide ligand which binds to a specific receptor and a positively charge peptide moiety with lysosomatic properties, useful in the delivery of a nucleic acid across a cellular membrane. The invention further relates to the use of these compounds for the delivery of a nucleic acid into the brain for diagnostic and therapeutic applications. BACKGROUND OF THE INVENTION [0003] The protective function of the blood-brain barrier becomes an important problem in the treatment of neurological diseases. The exclusion of blood-borne foreign substances also results in the exclusion of a large number of potentially therapeutic agents from the brain. The blood brain barrier (BBB) is a very complex endothelial interface separating the brain from the blood compartment and imped...

Claims

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Application Information

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IPC IPC(8): A61K38/00A61K48/00C07KC07K7/06C07K7/08C07K14/00C12N15/85
CPCA61K38/00C07K7/06C07K7/08C07K14/003
Inventor LAMENSDORF, ITSCHAKKATZHENDLER, JEHOSHUA
Owner LAMENSDORF ITSCHAK
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